Oregon Beach Monitoring Program: Bacterial exceedances in marine and freshwater creeks/outfall samples, October 2002-April 2005.

A total of 3086 samples, both marine (2916) and freshwater creeks/outfall samples (170) were collected and analyzed for enterococci during October 2002-April 2005, from 52 designated beaches in Oregon. A total of 3.2% (99/3086) of the samples at 21 beaches exceeded 158 most probable number (MPN)/100 milliliters (mL). The average enterococci levels of these 99 exceedances was 559 MPN/100 mL, with a maximum of 4352 MPN/100 mL (Otter Rock and Ona) and a minimum of 160 MPN/100 mL (Sunset Bay State Park, Bastendorff, and Mill). For marine water, 77/2916 (2.6%) exceeded 158 MPN/100 mL. For freshwater, 22/170 (12.9%) exceeded 158 MPN/100 mL, with a maximum of 587 MPN/100 mL at Sunset Bay. Sixty percent of the marine and 9% of freshwater exceedances occurred during the winter. Seventy-two percent (55/77) of the marine exceedances occurred after rainfall events (0.01-60.0 mm). At Harris and Mill Beaches, cumulative rainfall was highly correlated with bacterial densities, R = 0.7. Rainfall, at both beaches, explained about one-half the variation in log10 bacteria density (R2 = 0.5). Additional monitoring is warranted to further characterize bacterial contamination in Oregon waters.

Ultrase MT12 and Ultrase MT20 in the treatment of exocrine pancreatic insufficiency in cystic fibrosis: safety and efficacy.

BACKGROUND: Cystic fibrosis causes exocrine pancreatic insufficiency, leading to malabsorption. Supplemental pancreatic enzyme therapy alleviates the concomitant malnutrition experienced by cystic fibrosis patients. It is recognized that patients experience variations in clinical response to different brands of enzymes. This has prompted the US Food and Drug Administration to require that enzyme supplements be subjected to New Drug Applications. AIM: To investigate the safety and efficacy of supplemental pancreatic enzyme therapy in cystic fibrosis subjects. METHODS: We compared two doses of one formulation of enteric-coated pancreatic enzymes: Ultrase MT12 (12,000 lipase units per capsule) and Ultrase MT20 (20,000 lipase units per capsule), to placebo in two separate safety and efficacy studies. RESULTS: Mean total fat, protein and carbohydrate intake did not differ significantly between the groups. A significant difference in both fat and protein absorption occurred with the enzyme therapy groups. The Ultrase MT12 and Ultrase MT20 groups experienced a mean fat and protein absorption 79.4% and 83.8%, and 87.3% and 88.6%, respectively. No adverse events related to study drug were reported. CONCLUSIONS: This study further supports the use of enzymes to treat pancreatic insufficiency in cystic fibrosis. Excellent fat and protein absorption was achieved with minimal adverse events and safe doses.

Intervention strategies for children poorly adherent with asthma medications; one center's experience.

Poor adherence to medications and other aspects of the treatment plan is common in pediatric patients with asthma, and is a common reason for inadequate asthma control. In selected patients we have used electronic monitoring of pulmonary function, behavior contracts, home nursing visits, and medical neglect reports in an attempt to improve adherence and asthma control. Improved outcomes were seen with the most aggressive intervention, home nursing, and medical neglect referral, but not with less aggressive measures.

The effect of Cys LT1 receptor blockade on airway responses to allergen.

STUDY OBJECTIVE: To evaluate the effect of a potent experimental leukotriene receptor antagonist, MK-571, on airway responses to inhaled allergen. DESIGN: Randomized, double-blind, placebo-controlled, crossover trial. SETTING: Clinical research center. SUBJECTS: Eight male volunteers with allergic asthma. INTERVENTIONS: An intravenous loading dose was followed by an 8-hour infusion of MK-571 or placebo, with a 7- to 14-day washout between treatments. Allergen challenge was performed after the loading dose and a histamine challenge was performed before and 24 hours after allergen. MEASUREMENTS AND MAIN RESULTS: Forced expiratory volume in 1 second was measured serially. MK-571 provided about 50% protection during maximum early and late responses compared with placebo (p=0.005), but airway obstruction persisted 8-24 hours after allergen on both treatment days. Airway responsiveness to histamine was not significantly attenuated at 24 hours. CONCLUSION: Blocking Cys LT1 receptors for 8 hours attenuated the early and late responses but did not interrupt the cascade of events leading to subsequent allergen-induced airway obstruction and hyperreactivity.

The Schizosaccharomyces pombe hst4(+) gene is a SIR2 homologue with silencing and centromeric functions.

Although silencing is a significant form of transcriptional regulation, the functional and mechanistic limits of its conservation have not yet been established. We have identified the Schizosaccharomyces pombe hst4(+) gene as a member of the SIR2/HST silencing gene family that is defined in organisms ranging from bacteria to humans. hst4Delta mutants grow more slowly than wild-type cells and have abnormal morphology and fragmented DNA. Mutant strains show decreased silencing of reporter genes at both telomeres and centromeres. hst4(+) appears to be important for centromere function as well because mutants have elevated chromosome-loss rates and are sensitive to a microtubule-destabilizing drug. Consistent with a role in chromatin structure, Hst4p localizes to the nucleus and appears concentrated in the nucleolus. hst4Delta mutant phenotypes, including growth and silencing phenotypes, are similar to those of the Saccharomyces cerevisiae HSTs, and at a molecular level, hst4(+) is most similar to HST4. Furthermore, hst4(+) is a functional homologue of S. cerevisiae HST3 and HST4 in that overexpression of hst4(+) rescues the temperature-sensitivity and telomeric silencing defects of an hst3Delta hst4Delta double mutant. These results together demonstrate that a SIR-like silencing mechanism is conserved in the distantly related yeasts and is likely to be found in other organisms from prokaryotes to mammals.

The conserved core of a human SIR2 homologue functions in yeast silencing.

Silencing is a universal form of transcriptional regulation in which regions of the genome are reversibly inactivated by changes in chromatin structure. Sir2 (Silent Information Regulator) protein is unique among the silencing factors in Saccharomyces cerevisiae because it silences the rDNA as well as the silent mating-type loci and telomeres. Discovery of a gene family of Homologues of Sir Two (HSTs) in organisms from bacteria to humans suggests that SIR2's silencing mechanism might be conserved. The Sir2 and Hst proteins share a core domain, which includes two diagnostic sequence motifs of unknown function as well as four cysteines of a putative zinc finger. We demonstrate by mutational analyses that the conserved core and each of its motifs are essential for Sir2p silencing. Chimeras between Sir2p and a human Sir2 homologue (hSir2Ap) indicate that this human protein's core can substitute for that of Sir2p, implicating the core as a silencing domain. Immunofluorescence studies reveal partially disrupted localization, accounting for the yeast-human chimeras' ability to function at only a subset of Sir2p's target loci. Together, these results support a model for the involvement of distinct Sir2p-containing complexes in HM/telomeric and rDNA silencing and that HST family members, including the widely expressed hSir2A, may perform evolutionarily conserved functions.

Fatal asthma in children: a nurse managed model for prevention.

In response to the rise in pediatric asthma-related deaths and the absence of guidance in the literature for prevention of such deaths, a nurse managed program was designed to prevent death in children at risk for death from asthma. Published guidelines for identification of children with life-threatening asthma were used to develop a comprehensive health care network designed to provide early, aggressive medical attention and rapid access to care for selected children. The program has been successful in assuring more aggressive and timely intervention for asthma attacks and there have been no deaths from lack of access to appropriate care since the program's inception.

The Red Alert Program for life-threatening asthma.

OBJECTIVE: To design a program to prevent death in children with life-threatening asthma. METHODS: Using published guidelines to identify patients with life-threatening asthma, we developed a comprehensive medical network involving emergency medical services, school workers, extended family members, local physicians, emergency rooms, and a referral center. The network was designed to provide early, aggressive medical attention and rapid access to care for patients enrolled in the program. RESULTS: In 8 years of operation, 75 patients have been enrolled. There have been 270 hospitalizations, with life-threatening asthma occurring in 25 admissions in 15 patients. Three patients have died; of these, two did not use the services provided by the program. CONCLUSIONS: The Red Alert Program has been successful in assuring more aggressive and timely intervention for asthma attacks and seems to have prevented deaths. Patients and their families express a high degree of satisfaction with the services. The Red Alert Program may serve as a model for other programs for children with life-threatening asthma.

An uncertain silence.

Transcriptional silencing is the most well-studied epigenetic phenomenon in yeast. In Saccharomyces cerevisiae, silencing has recently been found at previously unidentified loci. In addition to the silent mating-type loci and telomeres, genes within the ribosomal DNA and, perhaps, at undefined aging loci are silenced. Efficiency of silencing at different loci varies and is affected by competition between the loci and by the involvement of different factors in distinct protein complexes. The recent discovery of conserved gene families encoding proteins related to modulators of acetylation and deacetylation suggests mechanisms for differential regulation of silencing at known loci and the existence of additional, as yet undiscovered, silenced loci.

Breaking the cycle: lidocaine therapy for habit cough.

Habit or psychogenic cough is a common problem of unknown etiology with a variety of suggested therapies. A postulated mechanism is a cough which causes vibration of the airway walls, irritation and more cough. Nebulized lidocaine breaks this cycle and allows control and resolution of the cough.

Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme.

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates. Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition. Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS. Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon. Reexamination of the identity set of tRNA(Gln) in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding. Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end. The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition. Furthermore, we show that E. coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA. The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases.

Functional connectivity between tRNA binding domains in glutaminyl-tRNA synthetase.

The structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) in complex with tRNAGln and ATP has identified a number a sequence-specific protein-tRNA interactions. The contribution to glutamine identity has previously been determined for the nucleotides in tRNAGln. Here, we report the mutational analysis of residues in all three tRNA recognition domains of GlnRS, thus completing a survey of the major sequence-specific contacts between GlnRS and tRNAGln. Specifically, we analyzed the GlnRS determinants involved in recognition of the anticodon which is essential for glutamine identity and in the communication of anticodon recognition to the acceptor binding domain in GlnRS. A combined in vivo and in vitro approach has demonstrated that Arg341, which makes a single sequence-specific hydrogen bond with U35 in the anticodon of tRNAGln, is involved in initial RNA recognition and is an important positive determinant for this base in both cognate and non- cognate tRNA contexts. However, Arg341, as well as Arg402, which interacts with G36 in the anticodon, are negative determinants for non-cognate nucleotides at their respective positions. Analysis of acceptor-anticodon binding double mutants and of a mutation of Glu323 in the loop-strand-helix connectivity subdomain in GlnRS has further implicated this domain in the functional communication of anticodon recognition. The better than expected activity (anticooperativity) of these double mutants has led us to propose an "anticodon-independent" mechanism, in which the removal of certain synthetase interactions with the anticodon eliminates structural constraints, thus allowing the relaxed specificity mutants in the acceptor binding domain ot make more productive interactions.

Aminoacyl-tRNA synthetases optimize both cognate tRNA recognition and discrimination against noncognate tRNAs.

Specific protein--nucleic acid interactions are usually the product of sequence-dependent hydrogen bonding. However, in the crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) in complex with tRNAGln, leucine 136 (Leu136) stabilizes the disruption of the weak first (U1-A72) base pair in tRNAGln by stacking between A72 and G2. We have demonstrated, by a combined in vivo and in vitro mutational analysis, that Leu136 is important for tRNA specificity despite making no hydrogen bonds with tRNAGln. Both more (L136F) and less (L136V, L136M, L136A, and L136T) mischarging mutants of GlnRS have been identified. GlnRS(L136F) is more mischarging and less specific than wild-type GlnRS in vivo, due not to an increased affinity for the noncognate tRNAs but to a decreased affinity for tRNAGln. Also, unlike other mischarging mutants of GlnRS that have been characterized, it does not exhibit generally relaxed tRNA specificity in vivo and mischarges only a subset of the tRNAs tested. A possible sequence preference for a Py1-Pu72/Pu2-Py71 combination is suggested. The L136A/M/T/V mutants are the first GlnRS variants, including wild-type, expressed on pBR322 which no longer mischarge tyrT(UAG) in vivo. We have shown that, while the L136A mutant is less mischarging than wild-type both in vivo and in vitro, it is not more specific as it also exhibits reduced affinity for its cognate glutamine tRNA. On the basis of these results, we suggest that the aminoacyl-tRNA synthetases have evolved to balance cognate tRNA recognition and discrimination against noncognate tRNAs.

The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability.

Genomic silencing is a fundamental mechanism of transcriptional regulation, yet little is known about conserved mechanisms of silencing. We report here the discovery of four Saccharomyces cerevisiae homologs of the SIR2 silencing gene (HSTs), as well as conservation of this gene family from bacteria to mammals. At least three HST genes can function in silencing; HST1 overexpression restores transcriptional silencing to a sir2 mutant and hst3 hst4 double mutants are defective in telomeric silencing. In addition, HST3 and HST4 together contribute to proper cell cycle progression, radiation resistance, and genomic stability, establishing new connections between silencing and these fundamental cellular processes.

Substrate selection by aminoacyl-tRNA synthetases.

The integration of genetic and biochemical approaches to study the crystal structure of the glutaminyl-tRNA synthetase (GlnRS):tRNA(Gln):ATP complex has elucidated the mechanism by which GlnRS selects its cognate tRNA for aminoacylation. Three principal types of interaction have been identified: interaction with specific bases in the cognate tRNA, rejection of non-cognate tRNAs, and activation of the active site upon cognate tRNA binding. The recent solving of the crystal structure of tryptophanyl-tRNA synthetase (TrpRS) has allowed comparable studies to be initiated in an aminoacyl-tRNA synthetase which, unlike GlnRS, does not require tRNA binding prior to amino acid activation.

Pediatric bronchography performed through the flexible bronchoscope.

Ten children, ranging in age from 2 months to 16 years, were evaluated by bronchograms performed during bronchoscopy. Nine of the ten children had significant pathology demonstrated by the bronchogram that elucidated findings unclear from other imaging procedures at the time of bronchoscopy. Despite the generally negative opinions about the utility of bronchograms in children, information obtained by bronchography can be quite useful. Bronchography during bronchoscopy proved to be quick, simple, and safe.

Selectivity and specificity in the recognition of tRNA by E coli glutaminyl-tRNA synthetase.

The specific recognition by Escherichia coli glutaminyl-tRNA synthetase (GlnRS) of tRNA(Gln) is mediated by extensive protein:RNA contacts and changes in the conformation of tRNA(Gln) when complexed with GlnRS. In vivo accuracy of aminoacylation depends on two factors: competition between synthetases, and the context and recognition of identity elements in the tRNA. The structure of the tRNA(Gln):GlnRS complex supports studies from amber and opal suppressor tRNAs, complemented by in vitro aminoacylation of the mutated tRNA transcripts, that the glutamine identity elements are located in the anticodon and acceptor stem of tRNA(Gln). Recognition of individual functional groups in tRNA, for example the 2-amino group of guanosine, is also evident from the result with inosine-substituted tRNAs. Communication between anticodon and acceptor stem recognition is indicated by mutants in GlnRS isolated by genetic selection with opal suppressor tRNAs which are altered in interactions with the inside of the L-shaped tRNA. We have also used genetic selection to obtain mutants of GlnRS altered in acceptor stem recognition with relaxed specificity for amber suppressor tRNAs, and a more extensive mutational analysis shows the importance of the acceptor binding domain to accurate recognition of tRNA.

The recognition of E. coli glutamine tRNA by glutaminyl-tRNA synthetase.

A variety of genetic, biochemical and structural studies have been used to determine factors ensuring the accuracy of recognition by aminoacyl-tRNA synthetases for tRNA. The identity elements of Escherichia coli tRNA(Gln) are located mainly in the anticodon and acceptor stem, and ensure the accurate recognition of the tRNA by glutaminyl-tRNA synthetase. We summarize a number of experimental techniques to define the accuracy of aminoacylation in vivo and in vitro.

Synthetase competition and tRNA context determine the in vivo identify of tRNA discriminator mutants.

The discriminator nucleotide (position 73) in tRNA has long been thought to play a role in tRNA identity as it is the only variable single-stranded nucleotide that is found near the site of aminoacylation. For this reason, a complete mutagenic analysis of the discriminator in three Escherichia coli amber suppressor tRNA backgrounds was undertaken; supE and supE-G1C72 glutamine tRNAs, gluA glutamate tRNA and supF tyrosine tRNA. The effect of mutation of the discriminator base on the identity of these tRNAs in vivo was assayed by N-terminal protein sequencing of E. coli dihydrofolate reductase, which is the product of suppression by the mutated amber suppressors, and confirmed by amino acid specific suppression experiments. In addition, suppressor efficiency assays were used to estimate the efficiency of aminoacylation in vivo. Our results indicate that the supE glutamine tRNA context can tolerate multiple mutations (including mutation of the discriminator and first base-pair) and still remain predominantly glutamine-accepting. Discriminator mutants of gluA glutamate tRNA exhibit increased and altered specificity probably due to the reduced ability of other synthetases to compete with glutamyl-tRNA synthetase. In the course of these experiments, a glutamate-specific mutant amber suppressor, gluA-A73, was created. Finally, in the case of supF tyrosine tRNA, the discriminator is an important identity element with partial to complete loss of tyrosine specificity resulting from mutation at this position. It is clear from these experiments that it may not be possible to assign a specific role in tRNA identity to the discriminator. The identity of a tRNA in vivo is determined by competition among aminoacyl-tRNA synthetases, which is in turn modulated by the nucleotide substitution as well as the tRNA context.